Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa

Bacteria have a broad array of defence mechanisms to fight bacteria-specific viruses (bacteriophages, phages) and other invading mobile genetic elements. Among those mechanisms, the ‘CRISPR-Cas’ (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR-associated) system keeps record of previous infections to prevent re-infection and thus provides acquired immunity. However, phages are not defenceless against CRISPR-based bacterial immunity. Indeed, they can escape CRISPR systems by encoding one or several anti-CRISPR (Acr) proteins. Acr proteins are among the earliest proteins produced upon phage infection, as they need to quickly inhibit CRISPR-Cas system before it can destroy phage genetic material. As a result, Acrs do not perfectly protect phage from the CRISPR-Cas system, and infection often fails. However, even if the infection fails, Acr can induce a lasting inactivation of the CRISPR-Cas system. The method presented here aims to assess the lasting CRISPR-Cas inhibition in Pseudomonas aeruginosa induced by Acr proteins by:• Infecting the P. aeruginosa strain with a phage carrying an acr gene.• Making the cell electrocompetent while eliminating the phage• Transforming the cells with a plasmid targeted by the CRISPR-Cas system and a non-targeted one to measure the relative transformation efficiency of the plasmids. This method can be adapted to measure which parameters influence Acr-induced immunosuppression in different culture conditions.


Introduction
The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats -CRISPR-associated) system is a bacterial defence system used to prevent invasion by Mobile Genetic Elements (MGEs), including bacteriophage (phages) [1] . CRISPR-Cas systems are classified in 2 classes, 6 types and 33 subtypes [2] . CRISPR-Cas mode of action relies on a three-step process (reviewed in [3] ). First of all, fragments (protospacers) from invading MGEs are inserted in one of the CRISPR arrays on the bacterial chromosome. Then, protospacers are expressed as CRISPR RNAs (crRNAs) and associate with Cas proteins to form a so-called 'surveillance complex'. Finally, during subsequent invasion from the same MGE, the surveillance complex will be able to recognise, bind and cleave the invading genetic material. The protospacers have a fixed length and can only be acquired from the MGE or bound by the surveillance complex if they are flanked by a Protospacer Adjacent Motif (PAM) [4] . Protospacer length and PAM sequences are specific to each CRISPR-Cas subtype.
However, phages are not defenceless against CRISPR-Cas targeting. They can carry anti-CRISPR genes ( acr ), which lead to the production of Acr proteins early upon infection [5] . Acr need to be produced in a timely manner to prevent the CRISPR-Cas system from cleaving the phage genetic material [6] . Therefore, Acr sometimes fails to protect phage from the CRISPR-Cas system [7] . However, they induce a lasting immunosuppression state [7 , 8] , which this protocol aims to measure.
This method relies on transformation of a targeted plasmid, carrying a protospacer recognised by the CRISPR-Cas system of interest, and a control non-targeted plasmid, without protospacer. We describe here the use of pHERD30T plasmid, a P BAD -based shuttle vector carrying a gentamicin resistance marker [9] . This plasmid was selected because it is a well characterised P. aeruginosa plasmid and it carries a resistance gene for gentamicin, to which P. aeruginosa PA14 is sensitive. The protocol can be adapted to other P. aeruginosa plasmids with a suitable resistance marker. The method was originally developed to measure immunosuppression induced by AcrIF1 and AcrIF4 on type I-F CRISPR-Cas system carried by Pseudomonas aeruginosa UCBPP-PA14 (PA14). We also describe how to adapt to other CRISPR-Cas system, by giving the example of type I-E CRISPR-Cas system carried by P. aeruginosa SMC4386.

Strains and materials
a. Plasmid, bacteria, and phage strains -pHERD30T plasmid.
-ATP (NEB, USA). Targeted plasmid construction a. Protospacer design In order to be recognised by the CRISPR-Cas system, the targeted plasmid needs to include a sequence matching one of the strain's spacers, flanked by the Protospacer Adjacent Motif (PAM). For the Pseudomonas P. aeruginosa PA14 strain, we used a protospacer matching spacer 1 of CRISPR array 2 of the type I-F CRISPR-Cas system [10] . The protospacer sequence was cloned in plasmid pHERD30T at the HindIII restriction site in the multiple cloning site. The sequence of interest in the plasmid was the following: HindIII restriction site Spacer sequence PAM HindIII restriction site The oligonucleotides designed for cloning contain the protospacer sequence flanked by the PAM sequence, surrounded by overhangs of the HindIII restriction site (overhangs in small caps, protospacer in capital letters, PAM underlined): When adapting this experiment to a new strain, we advise to design at least two different targeted plasmids, targeted by different spacers from different CRISPR arrays, and to test them to select plasmids with the lowest EOT (efficiency of transformation) on CRISPR immune bacteria (see "protospacer choice" section). Please note that sequence and position of the PAM depends on the CRISPR-Cas system type and that restriction sites used for cloning can be different than the one proposed here.
To adapt this method to P. aeruginosa SMC4386 type I-E CRISPR-Cas system, we selected and tested protospacers matching spacer 1 from the CRISPR array 1 and by spacers 1 and 7 from CRISPR array 2 (cr1sp1, cr2sp1 and cr2sp7, respectively). These protospacers were inserted between the HindIII and EcoRI restriction sites of pHERD30T multiple cloning site. The sequence of interest in the plasmid were the following:

Spacer
HindIII restriction site PAM Spacer sequence EcoRI restriction site The oligonucleotides designed for cloning contain the protospacer sequence flanked by the PAM sequence, surrounded by overhangs of the restriction site (overhangs in small caps, protospacer in capital letters, PAM underlined): -cr1sp1  • Prepare a culture of the chosen strain ( e.g., P. aeruginosa PA14 or SMC4386) in fresh LB medium; incubate for 12-18 hours at 37 °C, 180 rpm. • Tip : the amount of overnight culture depends on the number of targeted plasmids to test; prepare 3 mL of overnight culture for the non-targeted plasmid and 3 mL of overnight culture per targeted plasmid to be tested. For the SMC4386 protospacer tests, we used 12 mL of overnight culture. • Transfer overnight culture in 1.5 mL microcentrifuge tubes (2 tubes for the non-targeted plasmid and 2 tubes per targeted plasmid to be tested). For the SMC4386 protospacer tests, the culture was divided in 8 tubes. -Count the number of transformants for each plasmid.
-Select the targeted plasmid that displays the lowest transformation efficiency compared to pHERD30T. This plasmid is then referred as pHERD30T-targ. For the SMC4386 protospacer tests, the selected plasmid was pHERD30T-cr2sp1 ( Figure 1 ).
d. Plasmid stock production -Transform 50 ng of pHERD30T and pHERD30T-targ in commercially available chemically competent E. coli DH5 α (subcloning efficiency) as per manufacturer's instructions.
-Add 950 μL of LB to the transformation mix, incubate at 37 ˚C for 1h at 180 rpm.
-Plate 50 μL on LB agar supplemented with 30 μg/mL of gentamicin sulfate -Incubate the plates at 37 ˚C for 12 to 18h.
-Select one clone from each construction, add them to 5 mL of LB supplemented with 30 μg/mL of gentamicin sulfate in cell culture tubes. -Incubate at 37 ˚C, 180 rpm for 12 to 18h. -Transfer 500 μL of each overnight culture into 100 mL of LB supplemented with 30 μg/mL of gentamicin sulfate in a 500 mL glass flask. -Purify the plasmids with plasmid midiprep DNA isolation kit ( e.g. , Qiagen Plasmid Midi kit), according to the manufacturer's instructions. -Determine the concentration and purity of the purified plasmids using a UV spectrophotometer.

Infection
-Prepare cultures of the chosen P. aeruginosa strains, including a control strain without a functional CRISPR-Cas system ( e.g., P. aeruginosa PA14 and PA14 csy3::LacZ ) in fresh LB medium in a 250 mL glass flask; incubate for 12-18 hours at 37 °C, 180 rpm. -Tip : the amount of overnight culture needed depends on the number of phage treatments; prepare 20 mL plus 10 mL per phage treatment ( e.g. , 50 mL for 3 phage treatment with DMS3 mvir , DMS3 mvir -AcrIF1 and DMS3 mvir-AcrIF4 ). If the total volume exceeds 50 mL, use a 500 mL glass flask, or divide in two different 250 mL glass flask to allow sufficient oxygenation of the culture. -Determine the cell density through OD 600 measurement. -Tip : for P. aeruginosa PA14, a 1:5 dilution of stationary phase culture should reach OD 600 = 0.6, which represents a titre of 3.5 ×10 9 Colony Forming Units (CFUs)/mL in the undiluted culture. -Incubate all plates for 12-18 hours at 37 °C -Count the number of transformant of pHERD30T and pHER30T-targ for each tested condition.
The Relative Transformation Efficiency (RTE) is calculated as RTE = number of pHER30T-targ transformants/number of pHERD30T transformants. -Optional step: count the number of colonies and plaques on the bacteria and phage control plates to assess if the phage and bacteria concentrations are variable across the different tested conditions.

Method validation
In Landsberger et al. [7] (figure 5), this experiment was performed using the strain P. aeruginosa PA14 (carrying a functional type I-F CRISPR-Cas system) and its lytic phage DMS3 mvir ( a priori targeted by PA14 CRISPR-Cas system). The RTE of the BIM2 strain (carrying 3 spacers against DMS3 mvir ) was close to 10 −4 , thus demonstrating efficient targeting of the plasmid, while the RTE of PA14 csy3::LacZ was close to 1 as the latter strain is not immune against pHERD30T and pHERD30Ttarg transformation. The RTE of the BIM2 strain infected by DMS3 mvir-AcrIF1 was significantly higher than the RTE of non-infected cells. Moreover, infection by a phage lacking AcrIF1 did not disturb the RTE of this strain and none of the phage infection disturbed the RTE of the control strain. Therefore, the observed RTE increase of the BIM2 strain infected by DMS3 mvir-AcrIF1 is specific to the presence of both a functional CRISPR-Cas system in the host strain and an acr gene in the phage genome. This demonstrates that this protocol efficiently measures Acr-induced immunosuppression in P. aeruginosa .

Ethics statements
This protocol does not include human or animal subjects.

Data availability
Data will be made available on request.